USPTO Patents Application 09750990

Y.S. CHANG & ASSOCIATES

K.P.O.BOX 136. SEOUL 110: KOREA

Member I AIPPI A^AA^LES
Fax : (82-2) isS^JOT / 5969
Phone : 556- 568 -0461
Telex : 24928 (YSCHANG)

FILE COPY

rnstiii*

Kind of Protection : PATENT
Applicant : NOVO NORDISK A/S
Title

A Method of Preparing a Variant of
a Lipolytic Enzyme

End

Document fifed

Filing Date

Kor. Applit No.

X

Application

August 22, 1996

96-704602

Petition for Exam

X

Power ol Attorney

#fftt.

September 3, 1996

ft

Prtertty Oocactnl

Argument

MR*

Amendment

Printed Matter

Appeal

Tour Ref.Ko.

4153.204-KR, LiAS/KHgt

X

Our Ref.Ho.

DK-6P-0825

aas«>ui«<triKa)9ti»g5i36-%

BEST AVAILABLE COPY

NZAS-0018666

TRANSLATION

Please be advised that the following Application Number has been assigned to this case by the Korean
Industrial Property Office as below :

TO : Y.S.Chang, Attorney

Appln. Date : February 22, 1995
Applicant Novo Nordisk A/S

NOTICE OF APPLICATION NUMBER
(PCT)

Date: September 12, 1996
Appln. No.: KPA Mo. 96-704602

Filing Date of :
the Translation

August 22, 1996

Request for Exam.: (yes, no)
Request for Earlier LayingUpen : (yes, no)
Commissioner

Korean Industrial Property Office

Remarks

<"™*><«> ratuted «ly by r«mal Request. Des,gn or trademark apphc.t.on, ,re automatically mn>b«l sorting I. the fib,, dates.

Case

Official Fees for Requests for Examination

Patent Application

Basic fee : USS 96.00. Additional fee for each cfaim in excess of oneO) : USS 22.00

Utility Model Application

1 Base fee : USS < V0Q. Additional fee for each da an in nccts of onc<1 ) : USS 12.00

4 " rSjZl'lf^f ^ r,, * B ' f ^^11? " rt « i, ^. lion f «- sh»ll be ma* al an, Korean National Treason Bank in lh« of-
foal form requmd by then Korean Industnal Properly Office : one oT the receipts issued by the ban. should be attached
documents. AlMmfc , Postal Money Order in the required amount may reptac. the bank payment a^eceip?

5 ' K^^^^Z^^T a ' Ct "* '"^D-*"""*" Ctel SO..130/W or Application Department (Td. 5M-6079) of ,h.

6. (Address of the. Korean Industrial Property Office) 82J-I Yeotsamslong. Kangnanvko. Seoul. IJ5-7S4. Korea.

Official FiiJng
Certificate

<PCT«

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NZAS -001 8667

TRANSLATION

PAPER PIMM TO ARTICLES 201 aod 203 OF TI KOREAN PATENT W

Appl icant

Novo Nordisk A/S
Novo Alle
DK-2880 Bagsvaerd
Denmark

Attorney

Y. S. Chang
J. S. Jeong

Code of
Attorney

K020
K184

Inventor (s)

Allan Svendsen
lb Groth Clausen
Jens Sigurd Okkels
Marianne Thellersen

do Novo Nordisk A/S

Novo Alle, DK-2880 Bagsvaerd

Denmark

Title of
Invention

A Method of Preparing a Variant of a Lipolytic Enzyme

International
Appln. Number

International

PCT/DK95/00079 FN ing Date Feb. 22, 1995

NZAS-0018668

TKANSLATTON

Priority
Claim

Art. 54 of
the Patent
Law

Country

Kind of
Appln.

Rling
Date

Appln.
No.

Certified Document

attached

not

attached

Denmark

Patent

Feb. 22, 1994

0217/94

0

We are filing this Application pursuant to
Articles 201 and 203 of the Patent Law.

August 22, 1996

Y. S. Chang

Patent Attorney

J. S. Jeong

Patent Attorney

To : Commissioner

Korean Industrial Property Office

Attached Documents

1 . Paper and Translations of Specification, Claims,
Abstract and Drawings

2. Power of Attorney with its Translation (will be filed)

3 . Translation of Priority Document (will be filed)

4. Translation of Amendment Original, Duplicate

Application

basic fee

20 pages

W 20,000

additional fee

58 pages

W 40,600

Official

Filing

Charges

Priority Claiming fee

Priority
Application :
1 case

W 18,000

Request for Examination fee

claim

Total

W 78,600

Each 3 copies

Each 1 copy

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5. ^SMa&S^ ^y«ife ?i* _c|i.5o| H . lanuginosa 3^ DSM 4109
^ H. lanuginosa^] ^^.-¥-3 -fr£7V^ 3 4*9°] 714, 3Efe Rh. mucoid 5^,
a-c- C. antarcticaS] #7] E)aM|2] -n->M-*ll 0 ) 4.

"-ffW^ -g-<Hfe-, ^i+oj^sj ofnijn^ t V7l7V H. lanuginosa 3444
ofnl^xj. ol^ol^B), ^7] e]sMS| 3<H-E 70%

tf*^, (27B^ *M*1 *«»tt'*£3. #3). <4«l-c) 75%, 80%, 90%,

Hi^r 95% ^-§^°]JI, g«f«H o_SL #7| Jffi^-g-^olja ^/g:^

4**5) oH>n>tb A]<i 3Efe ^71 tf£**Kr DNA -Ht-t- 7|£3.

-i-Bl ul^eJjLElS. ^4 £>8SH*Rr DNA *\<&*K s\*tt ^Jt^fe ^SPS^S.*

H. lanuginosa elaMflsJ -8-3E*fl<|J ^ 5lfecfl t oj^o. o^^s-oj eja^oj

N- 9? C-££¥- *J.i| £fe #4°)] SH^ltfS] #7lS) ^7>, oVn]^AV

Ajlsj «|.i+oi^a| cf^. *$4M ©Wic* ^. 7 jsj

- 14 -

— — — " " ~ » —

NZAS-0018685

7 Wife 2)4^1- -y-jis^fe dna Hit ^«i^-o.s^ ^^^4.

#3 *3U***S.e1h s. H . lanuginosa

SKWKr DNA *J<gs] £^3Hr DNA sffe 33.$ 3.3

«MM 443 ^ fltK o^oj ^ Al€ ^ ^ #<H 5 x sscoJl

"lei ^HJL 20% if-ofoiH, 5 x m^*L 50mM °J*>M«. pH 68 3 5^
s| i<»M DNASJ -g-oJJoijA^ -40^011^ l^l^oj <q n]

£^3M?U, °1<>H ~40t°lH 18*|^**F 100//M ATP5L SLf^ ^<U$ -8-«M*l

a^SH^oi S^SMtJM- ^ sambrook §-(et al., 1989)

H. lanuginosa a]^]s] ^ej^o] HSM-g-^ ^4^^ ^<^£

il-l-'H Hudson §(et al., 1989W 7j£3 ^^.a ^fli^cf. ^a^o] 25 q.
**** ol. ^o, WH - ^ o) ^ HudsQn . (et

al., 1989H s]*f| 7) #3 44 Western Blotting lEfe #4 gefjg-g

•2- ^lUS-aflS.^ ^ DSM 4109 3E£ a ^-a>^5.«-e1 <g-|- ^ H
lanuginosa iWU nfl. ^^^i #S«<>l-fr£& «J o?]fe DNA *\<g £ $7}
° W!c * 2 1 - 27 - 56-64. 81-99, 83-100, 108-116. 145-147. 174, 202-213. 205-211,

- 15 -

NZAS-0018686

226-227, 246-259 I£fe 263-269 ^s)& qo\s. $ » ojs. o^j-jj.

Sj-fe DNA *J<g2) °1 TIM- Sfe ^ ^#4.

-5M s\^s\ DNA ^ oj-nJicAj a\<£°. zj-z]- SEQ ID Nos. 1 ^ 23.^3

^«#<>i, S- *l€£-*8:§:iS <2JL3}-fJ-fe DNA ^<I£- P . cepacia, P. alcaligenes, P.
pseudoalcaligens, P. mendocina P. putida^jlS ■?■-§■), p. syringae, P. aeroginosa
£fe P. fragi Pseudomonas sppM 5^-, B. subtilis SEfe B. pumilus BacilJus
spp.sj St- SEfe S. scabies Streptomyces sp.sj ^s.^ ^ #4.

S. tf7] f>4 <HS 3. ^f-°{ EP 218 272, EP

331 376 5 EP 407 225 °fl 7]<£& 44 Pseudomonas 2J4^I, Sfe WO 88/09367 °fl

71 #3 ^ ^4^3+3 -8-5.3 34*flii *r Si 4.
$711 ^ji*>7| 3*11 g. £>*s| ^o)^^ 4£s} AStf* 4-8-sM 7|^^4.

°l 4^. «t»*©J 96 ofin|.aHA>^. 1^0.3. 3}°-

Asp 96 Val SEfe D96V
Asp 96* £fe D96*

- 16 -

NZAS-0018687

Asp 96 ValLys Sfe D96VK

Asp 96 Val + Glu 87 Lys Sfe D96V + E87K

96 ^ 87 fl*HM z}-zj- <4^hea> £ «^ ^ sjAjos

D96V, N £fe
D96V afe D96N

3 <447M, <H£ ^3 8U «-«^7l ^.o,^.

tttf ^i£^ afe o|« ^ o Wil4 ^ R, N> A> C( E>

G, H, I, L, K, M, F, P, S, T, W, Y, V, £fe a eWtfaj o\v)±&

2. H. lanuginosa ^aMl^l l-^^ojg. o^H t$°\*t ^(^7] =8*1$
*fr)<*7| *a^«o| 4 5j OJ^ o,*,^ cc^. 4

S- ^^A^f #7]$ £o| DSM 4109sL^El <3-|- ^ oj- H

lanuginosa e]3H] lEfe n^sj -fr*^ ^ igo^ cf£°] ^

- 17 -

NZAS-0018688

S58. T64, S83. N94. K98, 1100, A121, E129, D167. R205, K237, 1252, P256 5^ G263.

A. C, Q, E, G, H. I, L, K, M. F, P. S. T. W, Y. V, DS.^ #7)7}
tfUfi /r Si-S-°] 3H4.

£ ^£ DSM 4i09SL-¥-3 £-g- ^ 31^ H. lanuginosa eJaMI sfe o|
e) 4^1^ 3.$. 3 o. Si <^ 7 )^ ofPi^ ^. 7 , L26 4^ ^-Aijjf^

<>Mt<ih ^ R, N, A. C. Q, E, G, H, I, K, M, F, P, S, T, W, Y, V. «\± Bj-M-ofl

5- ^JH <4€- 3°]^ l-^o} K46R, E57G, G61S, S83T,

S58F, D62C, T64R, I90F, G91A, N92H, N94I, N94K, L97M, K98I,
I100V, D102K, A121V, E129K, D167G, R205K, E210W, K237M, N259W,
I252L, D254W, P256T, G263A, L264Q afe T267W ^ ^o\s_

^^2J oj^^ oj^ofl 14= ^o|3flfe cf^-£] s.<3^o) S83T, N94T,
A121V. D167G. R205K ^s] ^q.^. o|f.o|^icJ-.

£ 32)3 i-<3*loj2] § o|#o| 3.^ afe WQ 92/05249, WO 94/

25577 3 WO 94/0154H #<a^o|^ <H>=34 oj 7 )o|| 3 0)3 f-<g$o|o] ^

2:^«;S. °l¥-<H*lfe SL H. lanuginosa 2)4^3} igojsflf. a^-^cf.
£ «"8S| 4« °<M<h8- #«g^o|^^jo]£ «mi ojijLoJ*]^ DSM 41095.

-r-tH 4= Slfe- H. lanuginosa 5fe zl%s] %-A}if{2\ SMcf:

- 18 -

NZAS-00 18689

N94K+D96A
S83T+N94K+D9 6N
E87K+D96V
E87K+G91A+D96A
N94K+F95L+D96H

Al 2 1 V+R2 0 5K+E2 1 OQ

F95C+D96N

G91S+L93V+F95C

E8 7K+G9 1A+D9 6R+I 1 0 OV

E87K+G91A

SB 3T+E8 7K+Q2 4 9R

SB 3T+E8 7K+W8 9G+G9 1 A+N9 4K+D9 6V
N7 3 D+S 8 5T+E8 7K+G9 1 A+N9 4 K+D9 4 A
E8 7 K+G9 1A+L9 3 1+N9 4 K+D9 6 A
D167G+E210V

N7 3 D+E87K+G9 1A+N9 4 1+D9 6G

S83T+E87K+G91A+N92H+N94K+D96M

E210W

E5 6T+D5 7 L+I 9 OF+D9 6L+E9 9K
E56R+D57L+V60M+D62N+S83T+D96P+D102E
D57G+N94K+K9 6L+L97M

E87K+G91A+D96R+I1O0V+E129K+K237M+I252L+P256T+G263A+L264Q

E56R+D57G+S58F+D62C+T64R+E87G+G91A+F95L+D96P+K98I+K237M
K46R+E56R+G61S

D102K

D167G

N7 3 D+E8 7K+G9 1A+N 9 4 I+D 9 6G

E210V

E210W

N 2 5 1 W+D2 5 4 W+T 2 6 7 W

S 8 3T+E8 7K+G 9 1A+N9 2 H+N9 4 K+D96M

E5 6R+1 9 0F+D9 6L+E9 9K

D57G+N94K+D96L+L97M

- 19 -

NZAS-00 18690

7l#€4. S°l*fl«- ^*Rr tfloVS) «£«8.g. *eU.fr«etAS|S.

<>1 -aAja)} 3-6°M

* ^i^fe *M*te* £$-3Hr ^l-8-*>^ 5.4: «§EjJofl^

^ 5A4.

^sl SH^g. DNA ^il-l- ^«HS|-te ^ tf)^^ 7fl^^-

DNA ^^^Tfl 4* Slfe «lEl«a SiJi, £<8^- ^

3M4. *|Ejfe o.s. ^sffe *|iH, ^ S^sl^l

£*8*Hr ^ sin, oisjaj 4^1 <3<^ 4*H*r ^U^t 4*

- 20 -

NZAS-0018691

-8-5l€ ^ 5S4. ^-SJ ^BfleM £ <g^s} So]^ o^j^- DNA

-H^sl *U>.f. ^l^^fe ^g-^ i^-S-^oj «U^, E. coUe] lac S-H^sH ^-fr*!^.
Streptomyces coelicolor 6 >7>Hf>J dagA ^-fr^*)-, "fl-if-^ WO 93/10249°fl

7l£€ Bacillus Ucheniformis or-^*^ -fr #*>-fr3*KamyL), Bacillus stearo-

thermophilus ^£ -fr^rSl ^-n-^KamyM), Bacillus amyloliquefaciens

o-oV^^^ls) ^-rr^KamyQ), Bacillus subtilis xylA ^ xylB -ir^Hf-SI ^
-fr3*W4. ^a>i- 3$ oj^ A . oiyzae TAKA

OrUeMl, Rhizomucor miehei «>is)-2. e. =SEflo)i4^, A. niger ^ a-OrU^I,
A. niger # <iHJ$ a-*Y£Q*. A. niger tfao} 0 ^^, Rhizomucor miehei 2js}-*l] t
A. oryzae ^^Hj^ =S.^|o|-^| f A. oryzae B.^\S-±l £i2fl°|Jg. oH^J^Sidi Sfe
A. nidulans °HS.o}d1 #3^*1-^ -if£€

°o v 3l5}-#fe DNA ^-a^ 2:^71-^71] "S^g ^eK^s)- -Mgs. ol-f^
4rS€ ^ 5U4.

^ms«ih m?y 4*3^ ^ dna *j<a-£ 3

itttt ^ ^fsj <4|fc p uci9, pACYC177, pUBllG. PE194,

pAMBl ^ pIJ702^ -^a]

*HH*r ««|7r*« J£a], oiH-s-o^ B . subtilis SLfe B. UcheniformisS^^a]

- 21 -

NZAS-0018692

4^4, 44^ amdS, argB, niaD ^ sC lk£r Aspergills -ii^X^KfiA]^ sI^dI*]
H-2.?]4)5- ^ £fe 4i4£ °fl#§4 WO 91/1724341 7)<£%

43£»fl 1^4 3H4fev««l"&4 ^M}&3-*\ 4«3 4444# A>-g-^ Hi),

-JM*MI € 4 1 Slfe 43L4-°fl4 «<H4fe 24 4#*]44.

a. *l3$«t£££- £^4-5-3. n*)3- *«T* 4-§-4fe ^4-?-fls.

«#43. °1 «l4-¥-flfe 4€- <*4-¥-*l BEfe MS. 41°11 4*B 4^4^4-2-, 4 oW
■¥•41* °J-J:^4fe DNA 4t4 *i$oi) s)s8 £4441 o|^<H^ S14.

-fr34 ^ 4^ <£%is}jL ol^l-a- ^5.^- ^iL» 3-8-4^r

44-5- #344 4*1] A>-§-^ 0| ^-O^ ^^^oflTlj £ Sl4(^-Jis.,

41 ##4, Sambrook et al. (1989)).
^4 ^4# £ U^4 DNA ^3:1- 1 £*i44'S'4 443. 4*44fc ^44

4-S-34. 4I£tt 34441^- <£Mr44*»H4 ^ dna =f-^4: £4*«
* tfJlSl-SHr £ £^4 DNA -T2l^-3- t SI4. U^^S. 43

DNA 4^4 -*fl£<JH14 43- #44?fl -8-444 <H4 «fl^ -B-444^ -#4^4.
^ 34*113.4 DNA ?2r§-4 *<B, 4*^4 4#3

*fla^°f| o]^ o^c). cfl*>0L5., 4£fe <^HM|i4 4€ #344

44°)1 4#« 44s ^^^1 4 s 5A4.

-H- T34 i-fr^l- ife «*24 4 3.^^ -JM44 >BS^ *r $44

4**4 4SL, «fl«-l-4 4444 3Lfe i#) 4£4 34

- 22 -

NZAS-0018693

WWW «=flfe

Bacillus subtilis, Bacillus licheniformis, Bacillus lentus.
Bacillus brevis. Bacillus stearothermophilus , Bacillus alkalo-
philus, Bacillus amyloliquefaciens, Bacillus coagulans ,
Bacillus circulans , Bacillus lautus. Bacillus megaterium , Ba-
cillus thuringiensis , HL Streptomyces lividans SE-fe- Streptomyces

murinus zl#<S-$3\ q*QeW. ^ £. coli »*ej)e)o}.oii}
*NeH3 S3*!** <$^°\ Q^Q^ ^#&°fl 2\n 3Efe x >iHlfe

212- -fr^l^fe -S-e|sf7fl^- Saccharomyces Schizosaccharomyces^ ofl^.-&c^
Saccharomyces cerevisiacS.-^El ^d^l *r Si 4. ^r^-S- Aspergillus**) ofll-t-^
Aspergillus oryzae, Aspergillus niger SEfe- Aspergillus nidulans°l}.-frejsf7fl -#^4.

«|°H *1|3L^ SEtfSrfe ^ $1*}.

Aspergillus *g 3j *]£ EP 238 023°f) 7|#3<H Slrf.

^°Jcfl, o) ^o)4^ S^SHH #7) 71^-^ Hj-S}- £,g- ^

mtf^jZ. a)1 5. H/EEfe »]MofH go]*^ 31 ^tf^ ^0.5. o]f-<H^4.
41 m-*«h7i ^sfl A >-8-€ **|sl ^^i*

-23-

MZAS-0018694

°1°H <>]■& HS-O^OLe)-^, ^ISHJ 3.2.v\s.-LilsH %• HS4£.a43}2l

UUW#«*. 4424*. 4***1 P H 7-13^ Ji4 nFJU^iS pH 8-11

•& £^4 44*1. £ 444 «io]^|^ ^«3^o.s. 44 £4*4 4£<y

&4. n 443.*! 434. <?Hi#€ «|4, Hfe £±4 t§^£) ^4

24*4 ^ 5tf4.

»l-*34 4ftt}"£ 4SS<H US 4,106,991 4,661.452(1:4 Novo Industri A/S°J)4
7]#£44 424jI <343. <>1 §o):S) ^)2) tg-^cfl ojsfl ^ 214.

4^.4 4xr ^V^l 1000-20000^1 *zK4flS!3-*lH.) 4§(#b]
PEQ); 16-50 4U3R:*lS ^fl* 4-§M$^ ^4*; <&S#o]
12-207flsj ^&«*|* 15-8071)4 41*344^ ^fl 7 J- 311= 4*MSHS

tta*; ^ <a-ia#; jqtf-S; 4##4 5Lt- 31 4- se^ei^ t= 0 jcf.

^<H?14. 44££44fe 4##oj aj-^oii 4^. *4-i-,

^ Sfe ^&i2£, gc- ^.g. ^7^0.5.4 #43*€ ^ 5U4.

4^ 2Li $34fc 4 £4=44 £ 244 $14.

Ji3.€ lit EP 238,2164 7Ba|^ nj-g). ^ fli}.

- 24 -

NZAS-00 18695

°i <r 514. 70% #o|ji o-30%fe ^l-g-ngs

a*** «m*i#si 7|««^£ -s-°i£^ (

>«fl^fe Ji-f- 0-50%4 °^Mf5vflolM(LAS), £4-^*113 £3LMHm(AOS),
^^•l-SMtttfa* **Mfij(AS) t ^s-i- 41^1 £sWe(AEOS g=fe AES).

2*1- tttt*si4H£<SAS), <ii-4-#s. *i»<kv "fliHitit-s., at eH^-tMh

S&fe 4¥2£ *«t-fc"« «-fr$4.

0-40%4 ^3* ^s.-aeflo]E(AEO £fe AE), ?J- = 4-*L#€ ^-^-^^^l e.,

3ctt**HH-a8Ms, tt«.#el*ajaAlJ&, 1^4 I'll "t^K! 4^1 <* 4*14-3

H»*<H WO 92/06154o)l 7ie«)4& *]o)&j *ltt4HM«

#1-44*1). ^34^ si^W, *t«HL 4**14*11, *A]tn, (o|*«o)

^/SLfe 4€- 2} 4*1) 3M4.

l-65%4 4*b HI 3; SEfe ^iisdol^ E^ S >-B|jo|e >

5Efe ^el?l«la(«l**<H HoechstS.-?-E]2) SKS-6) **WI* *r

Si4.

Hfe ^44tf4 **Mls. <>l*<>ia ^ si 4.

- 25 -

NZAS-0018696

cflfe- 9}S.^]vi^^s.^CMC), &3("l£*|a3£)(PVP), ^HH^SI-geJ^KPEG),

^Nxr 3-£^°>*3<1«$i4<>Ki(TAED) ^^^4^#iMHJE(N0BS)
^Sr'^-ft-** A«l %^^54 t 54^ aj.-g-ttS £fe 2£ H202

€#.2.3. ol^-<H^ 51^ i«i?fla- $-8-^ ^ Slcf. tfl°>o.S, «flS"&<H

ofl^eflH ^-aV -frs^-i- 4-8-*M °i^«}- 5 J ^ Sli. °»f"&<H WO

92/19709 ^ WO 92/19708°H 7]£& a>Sf ^ ^cf.

* * 3 a, afe tfs. 2*

(Af-g- ^^e|A) pHfe JL* °81-!-<H 7-1151

- 26 -

NZAS-0018697

(1) i**o.3. °1^<H*1^ WSL 60Gg/lSl ^JSL-i- £fe ^VSL3.*\ aM

«-*«a*#n.flo]s. mm 5.3. aw

7 - 12%

<g5L% ofl^.A^sl|o)E (ojH-S^ Ci 2 - 18 ^a# 1-2
EO) SEfe ^^^5flo)e H-i-s-o] c )

1-4%

EO)

5 - 9%

ft-a-'+Ef- (NazCOs)

14 - 20%

-8-*iH3 tK!:^ (Na20, 2SiC>2)

2-6%

*l-§-2]3= (NaAlSi0 4 )

15 - 22%

^te^M (Na2S0 4 )

0-6%

(CsHsNasO^/CACM

0 - 15%

(NaBOa HaO)

11 - 18%

TAED

2-6%

0-2%

f^*flW)S#<H ^\^/o\b_^s± -g-^tMlJPVP
PEG)

0 - . 3%

0.0001 - 0.1%

4sm^ )

0-5%

- 27 -

NZAS-0018698

(2) ^.<LS. °l-f-°l*|rr ^SL 600g/lS] £fe n*g^SL5L*] jQ*

o - 11%

zzj 0 il rtil ^1 T=r / -jl s Ala o 1 O

EO) SLfe «**S|«la. (<41-1-<H Cis-is)

1 - 3%

EO)

5-9%

(NaaCOs)

15 - 21%

ifM^ (NazO, 2Si02)

1 - 4%

(NaAlSi0 4 )

24 - 34%

*#M-.S.-§- (Na2S0 4 )

4 - 10%

( CjH^aaOj/CsHgO^)

0 - 15%

0-2%

^^(°«1-1-<H <£n#M3.*}-$: -g-^^sfl. PVP,
PEG)

1 - 6%

0.0001 - 0-1%

0 - 5%

- 28 -

NZAS-0018699

(3) 4-g-o.S. 600g/l4 ^UJE. 1- 2V^^-o.s.^ 2:^1 3 4*8

5

- 9%

EO)

7

- 14%

*ltf<I*8: «l¥-(4|*l-°l C16-22 *l*^!r)

1

- 3%

(NaaCOs)

10

- 17%

fftf^ (NaA 2Si02)

3

- 9%

*l-§-ejE (NaAlSi0 4 )

23

- 33%

3H±4^.f- (Na2S04)

0

- 4%

4*4ih+&g- (NaBCb.H20)

8

- 16%

TAED

c

2

8%

^sflolH (^l-f^ EDTMPA)

0

- 1%

0

- 2%

^*i]H»f-o1 £e|<SM=l*J-g- pyp,
PEG)

0

- 3%

0.0001

- 0.1%

0

- 5%

- 29 -

NZAS -00 18700

(4) olf-o^^^ ^o]5_ 600g/12l Jf^S-l £fe 4^^5.5.^

8

- 12%

EO)

i n
1U

- 25%

sMH+mi- (NaaCCb)

14

4. "0

-§-«8^ MS (N a2 0. 2Si02)

1

- 5%

*!-§■£] S. (NaAlSiO<)

25

- 35%

^M-JLl- (Na2S0 4 )

0

- 10%

0

- 2%

*«-aK«t«TB-<H ^N-S/ofa^* PVP,
PEG)

1

- 3%

0.0001

- 0 • 1%

0

- 5%

- 30 -

NZAS-0018701

(5) i}-§.o.3. ^^sfl^Ni^ir

15

i —

- 21%

ofi^-aeflo] e (<|tfo| Ck-is'S-s-I-, 7
EO i\r C12-15 sf-2-i-, 5 EO)

12

- 18%

3

- 13%

0

- 13%

8

- 18%

I — — — x_

*>

0

- 3%

^AflHtrl:^ PVP f PEG)

0

- 3%

(B4O7)

0

- 2%

0

- 3%

8

- 14%

0.0001

- 0.1%

0

- »

- 31 -

NZAS-0018702

(6) 4-1-^3. °l-¥-<H*1fe W=r-*.*\ "A^^^M:

15 - 21%

^■Si: <**<a*t°l2e Hf:^ C12-15 $3* f 7
EO BEfe C12-15 'S'-S^ 5 EO)

3 - 9%

3 - 10%

*)-§-5Je (NaAlSiO*)

14 - 22%

9 - 18%

0 - 2%

0 - 2%

(il-tH PEG, PVP)

0 - 3%

■fla* £-8- fMt 251 ; 3800

0 - 3%

0 - 5%

0.0001 - 0.1%

)

0 - 5%

- 32 -

NZAS-00 18703

5

- 10%

3

- 9%

0

- 3%

^3:MM (NazCQs)

5

- 10%

**W iKt^" (Na20, 2SiQ2)

1

- 4%

*l-§:3^ (NaAlSi0 4 )

20

- 40%

#^4*1- (Na2S0 4 )

2

- 8%

^-S-^^^l- (NaB03-H20)

12

- 18%

TAED

2

- 7%

3^*8 Ht*^ ^zfl^M^^ PEG)

1

- 5%

0.0001

- 0.1%

0

- 5%

- 33 -

NZAS-0018704

8

- 14%

5

- 11%

0

- 3%

(NibCOa)

4

- 10%

-§-«l)^ fl-^ (NazO, 2Si02)

1

- 4%

^14-21 S (NaAlSKW

30

- 50%

(Na2S0 4 )

3

- 11%

a] e ax». vj-ef. (CeHsNasO?)

5

- 12%

<^*H W**<H PVP, ^zS^Mal^ -g-f^*||
PEG)

1

- 5%

O.O001

- 0.1%

0

- 5%

- 34 -

NZAS -001 8705

6

- 12%

1

- 4%

2

- 6%

14

- 22%

(NaAlSKW

18

- 32%

(Na2S0 4 )

5

- 20%

A)HaA]- q^^. (CeHsNasOr)

3

- 8%

q-mi- (NaBCbilaO)

4

- 9%

#3 Ml***] NOBS SEfe TAED)

1

- 5%

0

- 2%

<°|#1-<H ^ PEG)

1

- 5%

0.0001

- 0.1%

0

- 5%

- 35 -

NZAS-00 18706

(10) °l^o)*lfe 4^3L^.

1 *€«.^i^s4- o is (^^.^

15

- 23%

I a^*T "I ^ 1 a™ |™ V"!! a^n W2-15 ii-^Lir,

g

- 9%

/ CAJ \_ ^12-15 a s f D iSL?/

3

0

- 3%

1

- 5%

5

- 10%

2

- 6%

(B4O7)

0

- 2%

0

- 1%

1

- 3%

2

- 5%

0.0001

- 0.1%

0

* 5%

- 36 -

NZAS-0018707

(11) 0) = ^^ ^AJ^

2

20 - 32%

oflS-^sflolE <<^§oJ Cl2 . 15 o j2€ . ? EQ

C12-15 5 EO)

6 - 12%

2 - 6%

8 - 14%

J2.51MM. (B4O7)

r - 3%

0 - 3%

3 - 8%

0.0001 - 0.1%

0 - 5%

- 37 -

NZAS-0018708

(12)4-S- ±3L °]*?o)7)±. mo]s. 600g/is? sj-^^S.^ 44

25

- 40%

oJ ^} oil ril H «J-?V^5lflolE Hpf)
^TJ a ™ — H — » S a — 1| 1 — ■ » 1 l /

1

- 10%

^^W^l" (NaaCOa)

8

- 25%

-8-*M iMiS (Na20, 2Si02)

5

- 15%

^Afv+H^. (Na2S0 4 )

0

- 5%

(NaAlSiOJ

15

- 28%

(NaBCb.4H20)

0

- 20%

*MI*1 (TAED ££fe NOBS)

0

- 5%

0. 0001

- 0.1%

(<^«1-<H $£L, ^3*1 )

0

- 3%

- 38 -

NZAS-0018709

(13) iHJ^-tii-lMlM 3" SEfe (Ck-m^^sHSS. i)-i2)

(14) t±£o_S. °lf-<H*lfe 6G0g/l^ JfB|^£t- ?Hr 4U#-2.S.>*1 *3*l|

(Cia-is) tf^fcsWs

9

- 15%

3

- 6%

1

- 5%

(NaAlSi0 4 )

10

- 20%

H»t:*l Hoechst^-?-^ SK56)

10

- 20%

tKM-S* (Na2C0 3 )

3

- 12%

&m tK>£ (Na 2 0, 2S1O2)

0

- 6%

4

- 8%

13

- 22%

TAED

3

- 8%

0

- 5%

0.0001

- 0.1%

0

- 5%

- 39 -

NZAS-0018710

0.00001-lmg(^ &± ^23- zfltfH cfl-g-^fe <&°-£- ^7f^ ^ Slc)-ji
*]7l 4)3 2<4#<fr -g-°)£^, »W£-9, IM-fr-U SE^ -fr<g£l

tl-fl-fr ™7) €-$34 44 4^ 4 <3 g&e)

*Sh>44°lS. ^s^AjeflolE., ^elfl-e^Aj^olE, oVd]^ l-^7>e^ Bflo] e,

- *M 43 44 ^±/Hf-^ SE^ <+4i~f}-<gsl £44« *r

- 41 -

NZAS-0018711

e^s^^ol^Ajoj-ipH^ e^a. S . 2 .o] :i A)o>Jpa^ ( clu^oliAlo^zA^ ^
^^2) Tflsfe TAED4 #eHf- e^o|.x)1^o|eo1c}.

£ "frlSj ^71 ^2] 4^1 i^l-S- S.±% W

+£*0* *]-g-SH °J:^3}-1 ^ 21^-
*l7) JLdb, 214 ?»|a|.2.S ^ef^, = s.Bfl°M|

■95.. £3*0. ^ f-S-^- ^ Xlcf.

EP 551670, EP 533239, WO 9303129, EP 507404, US 5141664

GB 2247025, EP 41428S, GB 2234980, EP 408278, GB 2228945

GB 2228944, EP 387063, EP 385521, EP 373851, EP 364260

EP 349314, EP 331370, EP 318279, EP 318204, GB 220431 9 '

EP 266904, US 5213706, EP S30870, CA 2006687, EP 481547

EP 337760, W0 93/14183, US 5223179, WO 93/06202, WO 93/05132

WO 92/19707, W0 92/09680, WO 92/08777, WO 92/06161

WO 92/06157, WO 92/06156, WO 91/13959, EP 399752, US 4941988

US 4908148.

42

NZAS-0018712

2) 3 *l EH Sfe -3-^4 SEfe #*t£<>13 e]4^] ^-^fe S.SL

^jg^^ 30t<>ll^ 21M«4 3°i^°Jt «fl°^cf.

4) 3* ^ElS «-4S.el ^cf.

5) ^Efls. e]j^4 £H*Hr #?laf7l ^
#ej» «*3«(2K P.VJL : #3* i« = 3 : 1), Brilliant green

<*M<$, 0.004%). lOOmM ILeli ^ojj pH 9 g EGTA(^#^£ 5mM)5.

6) 3. B] 4^31- u]a4W 2>£ofl cj|*fl 7j-i^ 5H*\
Dobanol™25-7 ^Bj jMHa:

41*9 tflsj] MHJ-§- flfc iaej^f 5)«W ^.ojjo) o.02%

Dobanol™25-7S. CJ o]^.^^| fe aJ.^^. ^al*>ji^ # 7 | 7|#£ afsj- cfl^-^
£*J<3-§- 4£*H

a) 3*11 el 4^1 -8-#*> *}-8-
#e>^nlc pYESHL-i- ^£*1|>H 20€*<£ 12M Ift^^ ^el^cf.
^sl- e!4^I <g-x -fi-*i*Hr #-9^<>l2l 3:?l(05mM MnCl 2 ATPs]
l/^-4*#<H Leung et al., 1989. #2)«MM PCR-§- *f-g-*H

«>1 ^elfe S-S-^ol sj-Jl PCR ^ &<a^°l7>

- 44 -

NZAS-0018713

=M4€ PCR ^MW4 4^ rfi^oji oi^aj ^2:^(Muhlrad et aL. 1992)

^ 41" ^^H ^ ^ 3 £4 R C oli4 ^^^«ofl £1*1) #1.3^4.
87Hs| <a4S #^-g- A|<gsj-Ai?iji tfioj 5J ^o) ^ 2-378S1 #<9£°m

4 4-8-44 77fls} 442134^ 10,000-140,000 #€■•§- ^-fi-«>>n *l)2:4$4.

b) ^£3*3 344 S^^HMlt 44

#^£4-^7])44(^4;a^#Bj]iL4.s.)fe. l-^^^l^-^l 44^ s€ .oJ) tfl-g-*^

-rft-zfl£4H(§)-i 44$ DNA 4t4 ^^°fl tfl.g-44 ^^4.

444, 143 #««4-fr*7(4*lte 4«£$. H>dj 7flAl^|4 pcr «v-§-od 4-§-«4.
344 PCR ^^s>2 £*fl4n 45. «|Eis. €§-44?!4.

PCR »>-§-°II 4-§-44 Aji- *JeH #^«oi^€ $.-4 * ws.^ 4-8-45L-3-

$4.

DNA 4ggfcfe ABI Dye Terminator Cycle Sequencing 31 44 4-g-^el
^"l^lifl ABI DNA a^ 373A* a^^-o.^ ^*8484.

<$2\q ^o\^2\ ^Aj

H. lanuginosa 44*1 -8-^4 ^ asJ4 oWlc«Haa) 91-97 3? 206-2114
°J44 #iiia4-frtt« 44«42l# ^714 4s. ^ »$1}°I|4 7 }^^ 44 ;goj 42:
*rSi4.

44^tf 91-97 ^ 206-211°l 44*,JtHI 51 AS. IHN#7l

^±$& 4*11 334594. ^4 91-97^ 21444 3^ ^44

-^4 206-21 l^r 4444 ±^r £44 #21 ^£4- 44^4.

- 45 -

NZAS-0018714

aa 91-97 bM J±eiBj(e] = * Ws* e| tftfsj 25^4 3*9 eM»elB]iiL

0 <M33 DobanoJ™ 25-7 12# £21*134. aa g^gyoj #o|^o|^.^ s «^
°<H33 141- >H 1^514.

37M 4# 83, 103, 145)* PCR 2%

*M 31* ^ 58*1^, S83TS1 PGR t^. ,goj

*11

5'

5

C

G

T

5

C

3/

T

7

A

A

8

G

5:

93%

A;

2.33%

c;

2-33%

G

and

2.33%

T

T

8

T

T

A/C

T

T

5

C

C

7

T

T

5

C

6:

93%

c;

2.33%

A;

2.33%

G

and

2.33%

T

T

8

T

T

8

A.

6

C/G

T

5

6

G

7:

93%

G;

2.33%

A;

2.33%

C

and

2.33%

T

5

6

G

7

G

A

8

A

A

6

T

C

8:

93%

T;

2.33%

A;

2.33%

C

and

2.33%

G

7

S. l: eJl-eM]®sl 91-97S] ^3^€ <9s]*J #3«°M a}~§.£ -g-2j Ji-fr #efl

- 47 -

NZAS-0018715

3. 2

59

60

61

62

63

64

65

67

69

71

72

I

II

II

III

IV

II

III

V

V

III
II

G91A

S83T
S83T

S83T

E87K
E87K

E87K
E87K

G91A

S83T

N94K

D96A

N94TC

D96N

N94K

D96N

D96V

D96V

N94K

D96N

D96V

N94K

F95L

D96H

N94K

F95L

D9 6H

D96V

N94K

D96N

S. 2: Aspergillus tftitffE) pAHLS. e*£MW j, # ^

#°)n -8-«8* 0,0^ #^^0] o.^- ej-oja^^o^ ^ ^

^ 0l ^ I S Ofe 0.01% Dobanol m 25-7<* SHJ<?} a^ofl ufo^ 0>J g

floi*** DMA /t\

Cd

59

I

60

II

61

II

62

III

63

IV

64

II

65

III

67

V

52/68

vt

53

vt

69

V

71

in

72

11

73

VI

Sc £r & !L " « !» - " 92

TCT CGT TCC ATA GAG AAC TGG ATC

A

A C

C

A
A

GGG AAT
C

C

c

c
c
c

c
c
c

NZAS-0018716

93 94 95 96 97 98 99 100 -103 -145
CTT AAC TTC GAC TTG AAA GAA ATA -ATT -CAT

59

I

G

G

C

60

II

G

G

A

61

II

G

G

A

62

III

T

63

IV

C

64 '

II

G

G

A

65

III

G

T

67

V

A

C

A

C

52/68

wt

53

wt

69

V

A

C

A

c

71

III

G

T

72

II

G

A

A

73

VI

A

2£ 91 2)- 9321 ^7)1- ins) c/T ^ T/GiL 51^)34.

, i- v, 3 0 fl S£ 91-97ofl>H2j ^zflAElHf- 93%sj *\A$i% 2.33%sj 37i|<4

0*1* 2

^o]^^ ojo} 3-1- AlSiSf7| ^ Aj~g.£ ^^jo) 7 |$^
D167G+E210V

5raM EGTA..O.m* Dobanol™? 5-7. 0.006% T.AS
E8 7K+G9 1A+L9 3 1+N9 4K+D96A

5tnM EGTA.0.O?% Dobanor >, 25-7

N7 3D+S8 5T+E8 7 K+G9 1 A+N94 K+D9 6 A

S83T+E87K+W89G+G91A+N94K+D96V

E87K+G91A+D96R+I100V

S83T+E87K+Q249R

E87K+G91A

- 49 -

NZAS-0018717

Aspergillus orvzae°flA| Humicola lanuginosa gJsMsJ

Humicola lanuginosa ^aflsj #3-^^- EP 305216°fl 7l#^cf. ^ Aspergillus
oryzaeafH mm «g g| s^-a. 7 1£$4. 4-8-3 ^ ^^}^ p960

■2-5. <3 1 3€4.

3'^ m*]m. 4**1 ffliS £1-5*4: P960-I- Nrul *

BamHI ^£±3. 34. 2?Hsj ^ aH°W #efiu)= pB R322S

*3 BamHI/Nhel W<\ Nhel Klenow *sM«M£ #3.^3-

a1?U zi^oji °-<y$ BamHI ^ Nhel *$lf- #-B-«Hr #4^n]s. pAO i^. ^j-g-
534 <*2S). *3 4»H^1 p960°sL^ BamHI/Xbal *£

\i3j-*H pAHL-l- £84 (^)13H.).

-f}-^* fc^o]* a>-§. € ^e^S- Nelson « Long, Analytical

Biochemistry (180, 147-151(1989)ofl 7|*«4. ' o]^^ PCR-lV-§-o|H 7^}^^
«r4S Sr^a|o S ^3 DNA-7r^ A^^ojg.*) #<3tMS ^

st-fe PCR (§eH4*9 e^s) 3fc* -*<*■§■ i#$4. PCR«^ tf£

iLsL^Ei, #<3«|o]«. ^h>^ DNA aJttiifcS It^sH ^^}9]H

#4^4^ ^ o| «^ o. ^ AH 5oIH 7]#€4

Humicola lanuginosa 5l a}-^3} N94K/D96A #et-^nls.oj ^-j£-g-

g-4^4H nAHT.Sl *1^ 3 f

#4^1 = pAHL-i- 4-1-3) 50^ #-§-£?r-^a| SphlS. -iiSSMsl

- 50 -

NZAS-0018718

cf: 50mM NaCl, lOmM ME)i-HCl, pH 7.9, lOmM MgCfe. ImM cjEji-E 1/ig
§J Sphis] 2&9\. 37t°JM 2*|£ -§-<£ £-§-

£1h£-§- *5%5L (hb}^_hcI5. 5§^o| s}^ pH 75)

3-fr* PGR Stftfol -fry

« °l 7« *1 * ■ (=A) : 5 » -TATTTCTTTCAAAGCGAACTTAAGATTC-

CCGAT-3 '

PCR 5:^1 (=B) : 5 • -GGTCATCCAGTCACTGAGACCCTCTACCTATTAA-
ATCGGC-3 '

PCR 2:>0 2 (=C) : 5 ' -CCATGGCTTTCACGGTGTCT-3 '
PCR ^ el *|) (=D) : 5 ' — GGTCATCCAGTCACTGAGAC— 3 *

a* 3ft** cf-§~§- IN^fe «*<H4 -a^Al^lcf: lOmM SBli-HCI, pH 8.3. 50
mM KC1. 1.5mM MgCfe, 0.001% 0.2mM dATP, 0.2mM dCTP, 0.2mM dGTP,

0-2mM TTP, 2.5 ^a(Taq) *bM2M|.

#31 1°IK lOOpmol A, lOOpmol 79*1*11 B * Ifmol #e}i°l- *H

law ^^^oq 7^ji 95t:o))^ 2^, 37T;«fl^ 2£ * 72t:ofl^i 3^5.

0.6pmol 2*1 151 a|a^ # * j ^ ^ f^o)^ *J*g lOO^sJ ^ 7 J o^sj. ^

- 51 -

NZAS-0018719

#7fl 2*1 -LV-S-^l-ofl, lOOpmol 7Ha)^i C ^ lOOpmol AW ^
*i>, 95TC-fl^ 2£, 37t*H 2£ * 721C<>1H 3£°iL o^o^ 2&*M#-§-
°1 *«*«»l-fr*' #tf ^ ^Tfl 3<LiL °]^o] 5 4

4. *W 50^ 24*94 «*± BamHI 5 BstXIsL ft*^* lOOmM
NaCl, 50mM JLEl^-HCl, pH 7.9, lOmM MgCfe, ImM DTT, 10^ s| BamHI 3 10
BstXL 2AW *fl 37^1 $4. 733bp BamHVBstXI *«*

nAHLSl

*« #4^1=. pAHL-l- #71 3&a«MW BamHI g BstXISL s

Wli^ ten**. •) ^6)1, #71

«a<M*U E. sobs- *t}%H$m 4iaii *V*«4: #34 £

^*A±44 #^n] H fl*«4.

ABI X|*7|. 373AOW DyeDeoxy™ Teiminater Cycle Sequencing

* PAHLG91A/N94K/D96AS «3«8«U. pAHL* *SW?!4.
-3*1*1 4

Humicola el Ada cj-^- ^olaH# ^gU]?]^ se^p}^ ^jg,
4£4 ^oJ^lfe 3ojH 7 i£3 ^g.^ 7^4. 5^

ol= oj§4 ^^o)i 7^1^ 47H^ «7l^4.

- 52 -

NZAS-0018720

PAHLS83T/N94K/D96A

pAHLE87K/D96V

PAHLE8 7K/ G9 1A/ D 9 6 A

pAHLN9 4 K/ F9 5L/ D9 6H

PAHLF95C/D96N

pAHLG91S/L93V/F95C

5 ' -ATTTCTTTCAAAGCGAACTTAAGATTCCCGA-

TCCAGTTCTCTATGGAACGAGTGCCACGGAAAGA-3 »

5-TATTTCTTTCAAAACGAAGTTAAGATTCCCGATCC-
AGTTCTTTATGGAACGAGA-3 '

5 ' -TATTTCTTTCAAAGCGAAGTTAAGATTAG CGATC-
CAGTTCTTTATGGAACGAGA-3 '

5 ' -TATTTCTTTCAAGTGCAACTTAAGATTCCCGAT-3 '

5 ' -TATTTCTTTCAAGTTACAGTTAAGATTCCC-3 '

5 ' -TATTTCTTTCAAGTCACAGTTAACATTAGAGATCC-'
AGTTCTC-3 *

pAHLE87K/G91A/L93I/N94K/D96A

5 ' -T ATTT CTTTC AAAG C G AA CTT AATATT AG CG AT C -

CAGTTCTTTATGGAACGAGA-3 ■
PAHLD167G 5 ' -ATATGAAAACACACCGATATCATACCC— 3 •

PAHLA121V 5 ' -CCTTAACGTATCAACTACAGACCTCCA-3 *

PAHLR2 05K/E210Q 5 * -GCTGTAACCGAATTGGCGCGGCGGGAGCTTAGGG-

ACAATATC-3 '
pAHLN73D/S85T/E87K/G91A/N94K/D96A

5 ' -TATTTCTTTCAAAG CG AACTTAAG ATTAGCG ATC -

CAGTTCTTTATAGTACGAGAGCCACGGAA-

AGAGAGGACGATCAATTTGTCCGTGTTGTCGAG-3 1
PAHLS83T/E87K/W89G/G91A/N94K/D96V

5 ' -TATTTCTTTCAAAACGAACTTAAGATTAGCGATA-

CCGTTCTTTATGGAACGAGTGCCACGGAAAGA-3 *
PAHLE87K/G91A/D96R/I100V

5 ' -GCAAATGTCATTAACTTCTTTC^TCTGAAGTTAA-

GATTAGCGATCCAGTTCTTTATGGAACGAGA-3 *

5 ' -CCCGATCCAGTTCTTTATGGAACGAGTGCCACGG-
AAAG A— 3 '

5 ' -GAAGTTAAGATTAGCGATCCAGTTCTTTATGGAA—
CGAGA-3 •

5 • -CCCGATCCAGTTCTTTATGGAACGAGTGCCACGG-
AAAGA-3 '

5 ' - CGG AATGTT AG GTCTGTTATTG CCGCC - 3 '

pAHI>S83T/E87K
pAHLE87K/G91A
PAHLS83T/E87K

PAHLQ249R
5

- 53

NZAS-0018721

l-e>iD)c pAHLD167G/E210V

pAHLAl 2 1V/R2 05K/ E2 1 OQ
^ PAHLS83T/E87K/Q24 9R

6

Aspergillus ejifr att -fl-AHHal ig>a
Aspergillus ory zapS j («ajat-

100mC£| YPD (Sherman et al.. Yeast Genetics^ Cold Spring Harbor Labora-

tory, 1981)1- A, oryzae ^l i^s 3#*}?U 24*Kr 3^0^.3 aj «])

5^r» *>] BH-S.H(miracloth)l- o^sL ^jr 2 0(M2] 0.6M MgS0 4 S. 41^1^.
15m«2) 1.2M MgS0 4> lOmM NaHzPO*. pH = 5.8<^^ $ £ 4.

120mg2] Novozym 234. batch 1687-§- fl-fr^fe iml s] ££°J}-§-
^7 r «.Vcf. 5ft***. lutS) ^mg/me BSA <A|nr> r -fr^ H 25)» *}7(-^j7 a, D ,
3*HW m 15 .25A|<J fro). ^ H

lOOmM h^^-hci (pH = 7.0)* ^$4. ^£ej- i000g°IW 15 *

Stf ^*1?U «<g€*ftl- M gS0 4 o S ^e, ^$4. 2^01

STC <L2M 10mM h^^-hq pH = 75 10mM CaC)2) ^

stcd ^^m^u m#A*]m. o| e^^cf. ai^os ^

0.2-lm«sl STC°1| ^^^l^icf.
100*4 MM 100**1 STC^el 5-25«S| p3SR2 Hynes et al.. Mol. «

- 54 -

NZAS-0018722

Cel. Biol. Vol. 3. No. 8, 1430-1439, 1983<d 8*H -f-i^n] c ^! ^aj-gj-^ ^

nidulans amdS ^^o(|a| 25-g- o.2mts\ 60%

PEG 4000 (BDH 29576), lOmM CaCl 2 5 lOmM J=el^-HCl (pH = 7.5)-£ 7}^jl ^
±m £WJL (2?!), <L3. 0.$5ml2\ ^?)$ -g^ $7l*U £%X[

m. 25£ tf*r*U. 2.50Q g oflA 1 i 5 £ ^j.

2m£2l 1.2M i3«).#o| ^^1*14. ^ ^ 10M

pH = 7.0, 3± m<L!L*\$\ lOraMs} oM]koH H $ °};^7|
20mM<q CsQ* S^o)^ m ^ si^CCove, Biochem. Biophys.

Acta 113 (1966) 51-56). 4-7<a •§•?> 37TJ4M »H<£$ **ll £*ff- 2S JL, tfg-

*r°fl £<y #5141- ^*M1 $4. o] UvHHU ijL^o, ^

A. orvzae »3] Bl4>t| -8-Al-atlal ai-fl

*7l$ f^ D |£f #71 ^A H cfl 7 ,£ € 44 ^ A^uk^^ ^
P3SR2421 *a*UMI 4$ ^ orv'zae IFO 4177SL *a*HMtl*h
£°1 <S4 ^4^°1<=4 p3SR2sl £^#3.

»fl 0 ^l?la, 5^4 44.* 4-g-#4. 3^4 ^ «««S. oMsoHEl

4-8-$ =r Slfe- *3««*|ft 2$ **eW«tk 3 <y ^.oj ypd°IH ^#43 ^
"Htf e14*« 1HH tn« 3.4^4. ^

3*8 ^*!$-*f|t- HmtJL 30X:^ 4<3 **V 200m£ FG4 «1)4(3% *7^.

3% ^-S^liEfl, 1% 4M NaOHS. 70° 3. p H)sL 1L ffiwJ-^^^A^

^*1*!4. •
7

£ »qjsl eluMl #o\*l\«\ ^

- 55

NZAS-0018723

4**M m 7l^# -fi-sMls. ofej-Bjov j,o - 4-§-§h SH^2tf#EM|B}(ME

RCK)-gr 0.3.4 *ll2:42i4.

344 pH 33*}" *r**M pH 7°1H £AJ42i4. %ys\

£?n 1: tfjt ^^-g- 4H4. p h» 7s. ^

■S-ov ^Al ? l7ll $4. ^-g-e]4n ^ 434.

2: =*3.u>SLaEH. 50mM E.^^-^EfloiE

#*«KpH7).2.3. sg^-i- o]« DEAE-H^*#(i>fl TM) 3-g-$4.

4)44:2. 280nnHH £^7}- 1 0.05OD Ji4 4 ^Tfl^cf.

57H^ * J?-4f. 4**M **«1*4 £3 ^^afl(o-o.5M NaCDS
- -§-3^4. ££*^-§- #*4fe S.-8-4.

5Lw •r4«MS* 7>^o.S.^ 0.8MS 2^$4. 0.8M #3.^ *W*<»?£3.

°12J TSK 4 Butyl-Toyopearl 650C £(Tosoh Corporation Japan«M

?<3W 3-8-34. 0.8M tfifc- oHmoUs S3 ^.g. f^jg. 3.0,

#tJ| 4: -34x8 3*4fe ^ -§--§- ^4^1- 2ms«>9 i3^7l 4*8 § s i«

tW. "^fr 50mM EeJi-44NM e £^°Ji(pH7)i> S. 45] nj<g.a. o]= 37^

^ Q 4)4JLi (£4m«l 4-8-34. <S=7-ns. i±t -g-434.

-£ ^-^Sl 4^*115) flolaflsl 4)3}^

- 56 -

NZAS-0018724

£- Hwmcok lanuginosa 34*11 ?H*flsj 4447-8- o^sj a ja^gin^ 3

i-^-ofl ^01 150d!S | u|oi^o|)^ ^^sj-acf. «1«>]51# 32}-

*«: ^ 4<>l«i| 3^>«1#

3*: »*I*W*67|^ 3£<3.5x 3.5cm)
^•8-: 100% ^, SL^ #400

•a^: Sudan redS. ^qg. sJ-^Bj-H <q o.75mg ^S/g). 70133. 7}<I3 6^5] e}H# 4

3**1 34. <H^°1 aj-g- ^ ^.g. ia-ojAi 75t«>IlAl 30^-

^-"S 7f'M^ft4. 4£ofl 3},^ ^.g. ^

LAS (Nansa 1169/P, 30% a.m.) 1.17g/«
AEO (Dobanol™ 25-7) 0.15g/ «

3t!-iKiL# i.25g/«

i.oog/i

*«4a# 0.45g/«

0 .15g/«

pH: 102

e)4*8*£: 34*9 V»4&2\ 0.075, 0.188, 0.375, 0.75 % 2.5mg

aI^V: 20-g-
30TC

^^7|: *a^r #°fM 15$

- 57 -

NZAS-0018725

S^«£(~20t. 30-50% RH)

*>*3-#-8-^<«i* e|^>3 ?H*1I4 €h8s; H . lanuginosa el^# «}^594.

c 0Ji

AR = ARjjcj ( t )

°l7i^ ar£ #*h>m

Kfe K 2 Sr ^tflSL^s) ^«>4 °j °. 44\S4.

^-iltl^l- 3]<+s}-a4. 4-1-4 q<>\# 7fl-3<y*H ?

Uv = Cwt/C (fl)
0.25mg/£S} o}4$ **«<Cwr)S £-8: 5J4

4 *M *Va!4.

1) 0.25mg/«°IM °im i4(AR. tM )f 4? (i)-fr ?H-SsfSi4.

2) 0.25mg/£°lH o^aj. ^ jg.-^s izfl^fe 4^. aj^.

C - ( K(*-.>«j) )2 (jjj)

3) >R^ti^-s- 4 (nit ?i^sa4.

4-l-s) fi. io)i 44>dl4.

58 -

NZAS-0018726

.2. 1

E87K+D96V

1.2

S83T+N94K+D96N

2.3

N94K+D96A

2.7

E87K+G91A+D96A

2.6

N94K+F95L+D96H

3.3

D167G+E210V

5.0

E87K+G91A+L93I+N94K+
D96A

1.3

E8 7K+G9 1A+D9 6R+I 10 OV

5.2

E87K+G91A

5.0

N7 3 D+E 8 7 K+ G 9 1A+N 9 4 I +
D96G

1.3

SB 3T+E8 7K+G9 1 A+N9 2H+
N94K+D96M

3.8

K46R+E56R+G61S

1.9

D102K

0.2

D167G

1

N7 3 D+E8~7 K+G 9 1A+
N94I+D9 6G

1.3

E210R

2.7

E210K

5.5

E210W

1

N251W+D254W+T267W

0.8

S83T+E87K+G91A+N92H+
N94K+D96M

3-8

E5 6R+1 9 0 F+D9 6L+E9 9K

4.8

D57G+N94K+D9 6L+L97M

1.9

- 59 -

NZAS-0018727

Muhlrad et al. , 1992, Yeast, Vol. 8, 79-82

Shimada, Y- et al. (1989). cDNA Molecular Cloning of Geotrichum
candidum Lipase. J. Biochem., 106, 383-388.

Yamaguchi, S. et al. (1991). Cloning and structure of the mono-
and diglycerol lipase-encoding gene from Penicillium
camembertii U-150. Gene 103, 61-67.

Hass, M.J. et al. (1991) . Cloning, expression and
characterization of a cDNA encoding a lipase from Rhizopus
delemar. Gene 109, 107-113.

Kugimiya, W. et al. (1992). Cloning and Sequences Analysis of
DNA encoding Rhizopus niveus Lipase. Biosci. Boitech. Biochem.
56, 716-719.

Dartois, V. et al. (1993) . Cloning, nucleotide sequence and
expression in Escherichia coli of a lipase gene from Bacillus
subtilis 168. Biochemica et Biophysica acta 1131, 253-260.

Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd
Ed., Cold Spring Harbor, 1989.

R.K. Saiki et al. , Science 239, pp. 487-491, 1988.

Beaucage and Caruthers, Tetrahedron Letters 22, pp. 1859-1869,
1981.

Matthes et al., The EMBO J. 3, pp. 801-805, 1984.

J.0. Deshler, (1992) A simple method for randomly mutating
cloned DNA fragments by using chemical mutagens and the
polymerase chain reaction. GATA 9(4).: 103-106

Leung et al., Technique, Vol. 1, No. 1, pp. 11-15, 1989

- 60 -

NZAS-0018728

Fowler et al., Molec. gen. Genet., 133,. pp. 179-191, 1974

Brady et al . , "A Serine Protease Triad Forms the Catalytic
Centre of a Triacylglycerol Lipase", Nature 343, 1990, pp. 767-
770, 1990.

Tilbeyrgh, H. van, Egloff, M.-P., Martinez, c, Rugani,N.,
Verger, R. and Cambillau (1993) Nature 362, p. 814-820.
Interf acial activation of the lipa^e-prolipase complex by mixed
micelles revealed by X-ray crystal lography*

Hudson et al., Practical Immunology, Third edition, Blackwell
Scientific Publications, 1989

Alber, T. and Kawasaki, G. , J.Mol.Appl. Genet l, 419-434 (1982)

- 61 -

0

NZAS-0018729

1 1 3- %

(I) H^iL

(i) tfi kiclia A/S

(ii) ^-^^21 ^1^-: ^l^aflaL4;2| afl^fe

(iii) 2

(iv) -H^lf^:

(A) =7^?]: icJi tac)A a a/ S

(B) ?1 2j: icS. <&efl

(C) u)-ni«iE

<E) M-ef:

(F) 2880

(v) ^7}±-#t «

(A) "Bsj| f^; Floppy disk

(B) %Trtf: IBM PC

(C) ^Aj^: PC-DOS/ MS-DOS

(D) ill^o): Patentln Release # 1.0, Version # 1.25

(vi) cflaloi/t})^^

(A) °)#: ^ 2fl , ElS o}^E 7 }aE.

(C) 4153.204-WO
(ix) 3 a:

(A) #5}: +45 4444 8888

(B) £3}-^: +45 4449 3256
(Of^i: 37304

(2) SEQ H>NO: 1°I) e))^ ^iL
(i) >H1 ^HJ:

(A) 918 ^7)^-

(B) -8-^: «a^V

(D) -3^
cDNA

(vi) ...

(A)-n-7]^J|: Humicola lanuginosa

(ix)

(A) «l*/n«>l: CDS

(B) 3*): 1..873

(C) o]: mat_^E]H

(D) 67..S73

(xi) *117)#: SEQ ID NO:l:

- 62 -

NZAS-0018730

ATG AGG AGC TCC CTT GTG CTG TTC TTT GTC TCT GCG TGG ACG GCC TTG 48
Met Arg Ser Ser Leu Val Leu Phe Phe Val Ser Ala Trp Thr Ala Leu
_20 -15 -10

GCC AGT CCT ATT CGT CGA GAG GTC TCG CAG GAT CTG TTT AAC GAG TTC 96
Ala Ser Pro lie Arg Arg Glu Val Ser Gin Asp Leu Phe Asn Gin Phe
-5 1 5 10

AAT CTC TTT GCA CAG TAT TCT GCA GCC GCA TAC TGC GGA AAA AAC AAT 144
Asn Leu Phe Ala Gin Tyr Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn
15 20 25

GAT GCC CCA GCT GGT ACA AAC ATT ACG TGC ACG GGA AAT GCC TGC CCC 192
Asp Ala Pro Ala Gly Thr Asn He Thr Cys Thr Gly Asn Ala Cys Pro
30 35 40

GAG GTA GAG AAG GCG GAT GCA ACG TTT CTC TAC TCG TTT GAA GAC TCT 240
Glu Val Glu Lys Ala Asp Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser
45 50 55

GGA GTG GGC GAT GTC ACC GGC TTC CTT GCT CTC GAC AAC ACG AAC AAA -288
Gly Val Gly Asp Val Thr Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys
60 65 70

TTG ATC GTC CTC TCT TTC CGT GGC TCT CGT TCC ATA GAG AAC TGG ATC 336
Leu He Val Leu Ser Phe Arg Gly Ser Arg Ser He Glu Asn Trp He
75 80 85 90

GGG AAT CTT AAC TTC GAC TTG AAA GAA ATA AAT GAC ATT TGC TCC GGC 384
Gly Asn Leu Asn Phe Asp Leu Lys Glu He Asn Asp He Cys Ser Gly
95 100 105

TGC AGG GGA CAT GAC

GGC

TTC

ACT TCG

TCC

TGG AGG

TCT

GTA

GCC

GAT

432

Cys Arg Gly His Asp

Gly

Phe

Thr Ser

Ser

Trp Arg

Ser

Val

Ala

Asp

110

115

120

ACG TTA AGG CAG AAG

GTG

GAG

GAT GCT

GTG

AGG GAG

CAT

CCC

GAC

TAT

480

Thr Leu Arg Gin Lys

Val

Glu

Asp Ala

Val

Arg Glu

His

Pro

Asp

Tyr

125

130

135

CGC GTG GTG TTT ACC

GGA

CAT

AGC TTG

GGT

GGT GCA

TTG

GCA

ACT

GTT

528

Arg Val Val Phe Thr

Gly

His

Ser Leu

Gly

Gly Ala

Leu

Ala

Thr

Val

140

145

150

GCC GGA GCA GAC CTG

CGT

GGA

AAT GGG

TAT

GAT ATC

GAC

GTG

TTT

TCA

576

Ala Gly Ala Asp Leu

Arg

Gly

Asn Gly

Tyr

Asp He

Asp Val

Phe

Ser

155

160

165

170

TAT GGC GCC CCC CGA

GTC

GGA

AAC AGG

GCT

TTT GCA

GAA

TTC

CTG

ACC

624

Tyr Gly Ala Pro Arg

Val

Gly

Asn Arg

Ala

Phe Ala

Glu

Phe

Leu

Thr

175

180

185

GTA CAG ACC GGC GGA

ACA

CTC

TAC CGC

ATT

ACC CAC

ACC

AAT

GAT

ATT

672

Val Gin Thr Gly Gly

Thr

Leu

Tyr Arg

He

Thr His

Thr

Asn

Asp

He

190

195

200

GTC CCT AGA CTC CCG

CCG

CGC

GAA TTC

GGT

TAC AGC

CAT

TCT

AGC

CCA

720

Val Pro Arg Leu Pro

Pro

Arg

Glu Phe

Gly

Tyr Ser

His

Ser

Ser

Pro

205

210

215

GAG TAC TGG ATC AAA

TCT

GGA

ACC CTT

GTC

CCC GTC

ACC

CGA

AAC

GAT

768

Glu Tyr Trp He Lys

Ser

Gly

Thr Leu

Val

Pro Val

Thr Arg

Asn

Asp

220

225

230

ATC GTG AAG ATA GAA

GGC

ATC

GAT GCC

ACC

GGC GGC

AAT

AAC

CAG

CCT

816

He Val Lys He Glu

Gly

He

Asp Ala

Thr

Gly Gly

Asn

Asn

Gin

Pro

235

240

245

250

AAC ATT CCG GAT ATC

CCT

GCG

CAC CTA

TGG

TAC TTC

GGG

TTA

ATT

GGG

864

- 63 -

NZAS-0018731

Asn lie Pro Asp lie Pro Ala His Leu Trp Tyr Phe Gly Leu lie Cly
255 260 265

ACA TGT CTT TAGTGGCCGG CGCGGCTGGG TCCGACTCTA CCGAGCTCGA GATCT 918
Thr CyB Leu

(2) SEQ ID NO: 2°fl ^

(i) *\<£*&

(A) zJo]: 291 oH^aV

(B) c.^; o^^

(ii)

<xi) ^<g 7 |^: SEQ ID N0'2:

Met Arg Ser Ser Leu Val Leu Phe Phe Val Ser Ala Trp Thr Ala Leu
-20 -15 -10

Ala Ser Pro He Arg Arg Glu Val Ser Gin Asp Leu Phe Asn Gin Phe
-5 15 10

Asn Leu Phe Ala Gin Tyr Ser Ala Ala Ala Tyr Cys Gly Lys Aan Asn
15 20 25

Asp Ala Pro Ala Gly Thr Asn He Thr Cys Thr Gly Asn Ala Cys Pro
30 35 40

Glu Val Glu Lys Ala Asp Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser
45 50 55

Gly Val Gly Asp Val Thr Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys
60 65 70

Leu He Val Leu Ser Phe Arg Gly Ser Arg Ser He Glu Asn Trp He
75 80 85 90

Gly Asn Leu Asn Phe Asp Leu Lys Glu He Asn Asp He Cya Ser Gly
95 100 105

Cys Arg Gly His Asp Gly Phe Thr Ser Ser Trp Arg Ser Val Ala Asp
110 115 120

Thr Leu Arg Gin Lye Val Glu Asp Ala Val Arg Glu His Pro Asp Tyr
125 130 135

Arg Val Val Phe Thr Gly His Ser Leu Gly Gly Ala Leu Ala Thr Val
140 145 150

Ala Gly Ala Asp Leu Arg Gly Asn Gly Tyr Asp He Asp Val Phe Ser
155 160 165 170

Tyr Gly Ala Pro Arg Val Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr
175 180 185

Val Gin Thr Gly Gly Thr Leu Tyr Arg lie Thr His Thr Asn Asp He
190 195 200

Val Pro Arg Leu Pro Pro Arg Glu Phe Gly Tyr Ser His Ser Ser Pro*
205 210 215

Glu Tyr Trp He Lys Ser Gly Thr Leu Val Pro Val Thr Arg Asn Aep
220 225 230

- 64 -

NZAS -00 18732

lie Val Lys He Glu Gly lie Asp Ala Thr Gly Gly Aan Asn Gin Pro
235 240 245 2 50

Asn lie Pro Asp He Pro Ala His Leu Trp Tyr Phe Gly Leu He Gly
255 260 265

Thr Cys Leu

- 65 -

NZAS-0018733

1. (a) S. tfjfcSWfe- DNA <$2\q t-<3>?H-5HM- <QSL?]n

(b) ^MHW #3) (a)^^ oj^ DNA ^t-i: a, ^

(c) a ^aStU&s* ujji.aH ^#o9 tfl*8 sj^g ^

4-8-, *8U*F«&S|H«l 4* 3^ pgr 34 1.^^6)0.^^ 4^05

4. * HW fl<>W, DNA H«4 #««o|3 DNA

*l-g-SRr tf£3H-fe DNA -H^* <asU itf<R=. S^^o ]€ DNA

2}^$ ^^Mlil- 333**fciL DNA ^<g-§- ^afl

jis

3* #3 os. ^ #3.

- 66 -

NZAS-0018734

7. 4 6*4 3^4S7f A. niger, A. oryzae 9? A. nidulans ^ Aspergillus
^4 -MIS, S. cereviciae Saccharomyces ^2] 4s©l 3}-§- ^.^0.3. s].^.

8. 4 6*4 flow, ^457> a3-<$Hsl eW«r3 >j £> cc^oj.
Bacillus subtilis, Bacillus licheniformis,

Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus ,
Bacillus alkalophilus , Bacillus amyloliquefaciens, Bacillus
coagulans, Bacillus circulans, Bacillus lautus , Bacillus
thuringiensis m^Streptomyces lividans m.^Streptomyces murinus
Bacillus #2} /Hs. t e. coli =L&~&<>$si\ ^

^-^SLS. **3.

9. 4 1*4 flow, #oj*H3 BH^j, #ol*^, tfol-fr^. «8^a

«a=^Jfil <«* 4-3€ iJHJ* ^ 5!-S: a^i ^

10. 4 9^1 floW. M]°l-8:^ 4-3^84x1 ^2^- o)]4^Eflo)E g»/SEfe -g-^l-a:^
^■a^^lfe LAS £fe «HJ£jf|)o]jEol ^SLS. Sffe *»}.

11. 4 1*4 5fl«H- J H. &4(c)°W 3^4£7}- ^>d*^ l-«a^o|^^5| (

.12. 4 1* 11**21 o^ $ *4 fl«>W ( ojo}^ ..#^«oJ*«-ol JE.

13. 41* 12***1 012= « *4 flow, £ ^^^4^7} Hjal-4, 4^42*4,
^e|l}4 sfe i^se)44?l 3-§- ^Jos **j.

14. 412* £fe 13*4 floW. £ ^]^^-^l3L4i7f EjaMWs ^i^^ °Js}3} f-o*

- 67 -

NZAS-0018735

15. 4l4*J"°II SWA! °JS}^I #«« : <>1-S-«ol S].£ JjLo^ oj-^^j.- DNA

A ^ iZ.eJsMI3 ^e}^I #2} £ fe 2]j= « «}

±^ ^ f-sj -¥-^-o|i>H ^«gs|fe 3# «RJ *te «g-«.

16. *9 1*J- *fl*l 13*Jf^ o\± *j.oa saoj^-a ^^^^71- h14«-sj?leI

17. 418^-oH floH, 2. ^^#.4Jti7> 3***1 ^ 3^ £ 7 jo £

. ^

18. *lmH flo^, sl xMttqx^ ^S^fe DNA ^<go] Humicola sp.,
Rhizomucor sp., Rhizopus sp., Candida sp.s) 5^S.-?-e| ^ o{^. ^.g.

19. *I]18*<M1 Sa°H, 5L ^l^^-8ilJLi7> SlDf-^olj! 2.^^11. oJ-jL^-Sj-fe DNA
Wo] H. lanuginosa ofl^^, H. lanuginosa 3^ DSM 4109, Rh. mucoid

Sfe C. antarctica^ S^S.-?-^ -ff-S^ <r $!-§• tjj-^.

20. -JSiai ?o ?)fe dna.aUM D SM 4109£^e)
^ Slfe- H. lanuginosa el^^al 4°)^^ &\ 21-27, 56-64. 81-99, 108-116,
145-147, 174. 202-213, 226-227. 246-259 263-2694] ^3^- 3^5:
^ *-ir ^ASNHs-' ^-l- MlxJ 0.5. SHr .

- 68 -

NZAS-0018736

23. ftoM. a ^^J^f. <#313Ht DNA R cepada ^
P. alcaligenes, P. pseudoalcaligens 3Efe P. fragi Pseudomonas spp.2|
HLfe Bacillus^! 5^°1H -S-il ^ SKr f^|o S . ^ bj.*}

24. *8 1^ ifl*l 23*^3 oj^ $ aj-g. 0).= Hj-^oq SjsJI ^1^^*112:4:2)
$°1*11.

25. ^24*J°I| cf-g-S) ?)*1^ 3}oj£ ^oll/.^ S-ggoJs. o]^.of*]£. DSM
4109S.-?-e1 <g -g^ ^ 5Zt H. lanuginosa, 2j*Hl 5E^ O.S) ^}^2] •go^o] ^

K46, E56, S58, G61, T64, N73, S83, 190, G91, N92 , N94 , D96, L97,
K98, E99, 1100, D102, A121, E129, D167, R205, £210, K237, N251,'
1252, D254, P256, G263, L264£EfeT267.

26. oHic-iV #7] 56-64, 83-100 Sife 205-21H Sjafl 3<H.SL ^

^ DSM 41095L^-el ^ SAfe H. lanuginosa

27. >926^-ofl SW^, cf^-s] #<a^o|^ ajojs ^43. o]^.oj^ fe s * s.3 0.3.
*Hr 3°1*)|:

K46R, D57G, S58F, G61S, D62C, T64R, S83T, I90P, G91A,N92H, N94I,
N94K, L97M, K98I, I100V, D102K, A121V, E129K, D167G, R205K,'
E210W, K237M, N259W, I252L, D254W, P256T, G263A, L264Q Si^T267W.'

28. 4-l-sl #«a^ol^ ajoJiE. o]^^^]^ DSM 41095.^-El ^ Sife
H. lanuginosa el 4^1 JEfe ns} -S-Al-jiflo) Blojjif):

E87K+D96V
. E87K+G91A+D96A
N94K+F95L+D96H
A121V+R205K+E210Q
F95C+D96N
G91S+L93V+F95C
E8 7 K+G9 1 A+D9 6R+1 1 0 0 V
E87K+G91A

- 69 -

NZAS-0018737

S83T+E87K+Q249R

S 8 3T+E8 7 K+W8 9 G+G9 1-A+N9 4 K+D9 6V
N73D+S85T+E87K+G91A+N94K+D94A
E8 7K+G9 1 A+L9 3 1+N9 4 K+D9 6 A
D167G+E210V

N73D+E87K+G91A+N94I+D96G

S 8 3T+E8 7K+G9 1A+N9 2H+N9 4 K+D9 6M

E210W

E56T+D57L+I90F+D96L+E99K

E56R+D57L+V60M+D62N+S83T+D96P+D102E

D57G+N94K+K96L+L97M

E87K+G91A+D96R+I100V+E129K+K237M+I252L+P256T+G263A+L264Q

E56R+D57G+S58F+D62C+T64R+E87G+G91A+F95L+D96P+K98I+K237M
K46R,E56R,G61S

D102K

D167G

N73D+E87K+G91A+N94I+D96G

E210V

E210W

N2 5 1W+D2 5 4 W+T2 67 W
S83T+E87K+G91A+N92H+N94K+D9 6M
E56R+I9 0F+D9 6L+E9 9K
D57G+N9 4 K+D9 6L+L9 7M .

2- tfl^srH tfl^fl */3Efe

" #<3*M€ DNA ^l^ol^lfe DNA ^t-.

30. *24* Hl^l 28**3 «4^fe H. lanuginosa *J*M1 ° D >Jt^
*Hr ^8.2.3. ^ DNA

31. ^129^ 5E^ 30*<H| 4^ DNA

32. *J311WI Sl^^M, #e}^]tz tc^ ^BflEl^nj-^o] 3}^ ^ ^

- 70 -

NZAS-0018738

34. *]29*J- 30*<H <4a^ DNA ife »|31^ vfl*l 33H^sl <H^ *8"§r

35. *834*<W 5&oH, D 1^§1- $!-!- ^*J£.£ *^

36. *fl35*J4| 5A<H^, a* £fe «MeK ^3 #-§■ ^.2.3. t*$= xfla.

37. ^36^°fl 21 "H^M, A. niger, A. oryzae ^ A. nidulans Aspergillus #2}
Sfe S. cereviciae ^xr Saccharomyces #2] aJ|3E.o] s.^ gj.-

38. *836*<H 5a°H, a#-<?H^ «MeK ^ ofl##<H,

Bacillus subtilis, Bacillus licheniformis , Bacillus lentus,
Bacillus brevis, Bacillus stearothermophilus , Bacillus
eilkalophilus , Bacillus amyloliquefaciens, Bacillus cbagulans,
Bacillus circulans, Bacillus lautus. Bacillus thuringiensis S.^
Streptomyces lividans se^- Streptomyces murinus

Bacillus ^sj E. coli £-8- 3«-~g-^s] °\

39. *ll 1^ Ml*] 23*^s) #31 *8-^ofl ^o}sj) ^l^^iil- 4^3.

5L *ll-g-*l|:§L:fcsl- ^#°H tfl«j)

40. HHI^W 2.^s).oji^ ^34^- m*] 38*J-^2) ^-ofl 4§.
i ^l^^-aflJt^ HissH §#oj| rj|«fl >i]^|i4 *fl*S

41. *{|391h SEfe- 40*«W ".Wl s|St| ^2:^ ^l^§-«H2l±2l >S°l*t

- 71 -

NZAS -001 8739

42. <3s]3- h]-£^ S**$ afe J££€ S.±s] ^24^-
28*^ SEfe 41*8^21 ojH nj-^ ^l^^iiS] ^-^js. o|ijLoJ*] fe 4^

43. ^142*^ &<>H, ^Wg^ "E^^ 0.02-200mg-§- ^-frS}^ ^%<i.3.

44. *H42*3- 5Efe 43^°l) =ssJK*. <>Hm*i si^W. ^^4>9, 21431

HI-EM] ^7}S. 3-§- a,jo S .^ ^

45. *)124*J- 28*<HySj ^ ^- SEfe 41*^°fl *1^£*11:§L4iS} £o]*|}iL

46. *945*J-°fl ^7>S. HSeUoV^I, oVg^, Bj^tfj^ ^E^], 434.^ ^/

- 72 -

NZAS-0018740

DMA ^ff U^l^. ^ (c) S. ^l^Sfla:^ Bl5L«M zj-^ofl rfl^fl

^^r°fl tfl«fl 7fl#3 §<S^°1€

- 73 -

NZAS-0018741

NZAS -001 8742

NZAS-0018743

NZAS-0018744

^°I€V .3® ins. KEDEei 'ffia-

t 83&°li taste 3S7J PO?

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aft £t . 9821 c je|

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PCRQISI

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Fig. 4

5-4-

NZAS-0018745

5'
3'

3711 1

5'

■miiinmiCX

5'

5'

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5-5

NZAS-0018746

TRANSLATION

AMENDMENT

(Under Art. 205(1) of the Law)

IIOLUC

Novo Nordisk A/S

Filed by

Relation
to the case

Applicant

Nationality

Denmark

Address

NovoAlte

DK-2880 Bagsvaerd, Denmark

Attorney

Y. S.

Chang & J.

S. Jeong

International

Application

Number

PCT/DK95/00079

International

Application

Date

February 22, 1 995

Title of
Invention

A Method of Preparing a Variant of a Lipolytic Enzyme

International
Filing Date of
Amendment

- April 19, 1996

We are filing this Amendment pursuant to Article 1 1 5 of the Enforcement
Regulation of the Korean Patent Law.

Date : August 22, 1996

To Commissioner

Korean Industrial Property Office

Y. S. Chang
Patent Attorney
J. S. Jeong
Patent Attorney

Attached
Document

1 . Translation of Amendment

Original, Duplicate Each 1 copy

NZAS-0018747

°J3

•8- 4

-a >«+ #

^ 2]

AM?]

$iM*> =3- -§- 3 ?!

K020. K184

>H^-^1a] 824-20

^! J[ ^ i

556-8224-6

PCT/DK95/00079

1995\1 2*1 22H

\3 ^ <g

1996. 4.

19.

1996\1 8€ 22^

cj]2)°l # -g- ^

tflsj*] ^^ A > ^ ^

1. 113*1 *fl#>H

^ 1*

TEL 556-8224 -6

NZAS-0018748

1. (a) S. ^]^e«82Li# DNA Hl-i- °J^^ §<d£ 0 l-rr^-§- 1J.2-?M

(b) ^4i«fl>H #31 (a)°fH 1-^^<>1€ DNA ^H?)JL, $

(c) tR*|| s^-* l-£3'ol€ ^-g-^&^S ^

-fHJ-aflsJ Aj-g-, -§-e) 52.^-#e1| iE)HS) 4-8- 5t- PGR ^^WW
sin -a^slfe 3J-3- ^ wj.^

4. *ll 3*J-<fl 5fl'«>W, l-ol^ol-S-^-^fe Xf-AV, UV i4. N-^-N'-
Meg.-N-ulESdb^oW^dVlNNG). 0-WIl&lS^o].oi i o^xv, o?l^^|^i

5. *i| Si«H^, 1-^^»1€ DNA *HS£ l-'S^ol^ DNA 41sj
*l-§-*Hr #3L3HsHr DNA Alt^ ^SJJL 3 i^-sj-^ l^o]^ DNA
-M^-fr £fe f?i«!o|S DNA <H«-g-'*«*M

5^

6. J<| SH4, 1^£<>1£ DNA 41-1- A>-g.£ 3^4|3L7}

- 66 -

NZAS-0018749

7. 4 6*4 &°H, ^4i7V 3^- yM)ej<>> ^^e] Aflioi ^.g. s ? jc, s ^j.-

a 4 7*4 Sl<H A 1. <SHr43£7r A. niger, A. oryzae 3? A. nidulans Aspergillus
•MIS, SE^ S. cereviciae Saccharomyces 4£o] ^-§- s?jo.s

*U..

9. 4 7*4 5ft°W, ^4S7> ^4hMs) S^fil 41, 4«H,
Bacillus subtilis, Bacillus liche/iiformis,

Bacillus lentus, Bacillus brevis , Bacillus st&arothermophilus ,
Bacillus alkalophilus , Bacillus amyloliquefaciens, Bacillus
coagulans, Bacillus circulans , Bacillus lautus, Bacillus
thuringiensis SE^Streptomyces lividans 3.^Streptomyces murinus

*fr Bacillus ^s) e. coli HSr 3.^— §-^sl ^43 »r 4£°1

10. 4.2*4 sa«H-M. 43£*l£:iifc hW-&^, *oi*^, o<M£^,

£tt 4^*^44 44l€ Ml^-g- £fe *fe

*u.

11. 410*4 Stf<>H, 4«*Mi4fe ttst-fr 4*4l4 0 l£. -g-o]^
43t*34fe LAS Efe <y-^#5)l°lM<y 3}-§- a^o a *|.- *»J.

12. 4 1*4 a«H4, fc4(c)4*l. Aael^S ^4£?> ^«i^s| #«a^o|^-«v^ Bii
4^3el^, 4£e] 4€3.^-§- *4 ^fe *rfe *<3.

13. 4 1* Mi^l 12*f^sl *v sj-oj) ojo^, ojojaj i-<3<a°)-frlM a xi^-g-sn
ail- °a v i^5]-fe DNA *l£s| ^0.5. 5f±S^fe 4^-5.5.

14. 41* 13*^21 o]± sjofl flo)^, i 43 €-4 ££7} e)^4. 4^4e}-4,

- 67 -

NZAS-0018750

15. *H13*J £fe fl°H, ^ ^±.$# °}s\q

16. 415**1 Sl°i A i, -9*1*1 *««<>l*tf<»l H^H DNA

Wtt £Bl^^ i^s] #sj ^71 bJsl

17. 4 1*J" ^fl^l 16***1 <Hi^ $ ^-ofl <&o]Ai, s. *| 3**11 "I^S.-?^

18. *117^H fl<>W, £ *13€-*H£:fc7} S^ofl^ ^ alfe ^3^3.

19. SZ°H, 3- ^]^ns.±% 'tfiiW-te DNA Humicola sp.,
Rhizomucor sp., Rhizopus sp., Candida sp.s) Sf-S.-?-E| -ft-3E.fi ^ Sflfe 3i-t-

20. 419*J-^ £ ^fcsjji^ e|»l-* 0 U a^flMIl- "y-Jtsj-sHr DNA

H. lanuginosa <^##^. H. lanuginosa ^ DSM 4109, Rh. mucoid
tt, Sfe C. antarcticaSl Z^rS-^-i] -ft-£.f) ^ Sife ^ AS. *}fe tfU.

21. *fl20*J->Q Sa<H^, ^3 *|a«olt- U-^lfe DNA AJfc©] DSM 41093.-¥-iH

t Slfe H. lanuginosa el oV^^xv # 7 1 21-27, 56-64, 81-99. 108-116,

145-147, 174, 202-213, 226-227. 246-259 £fe 263-269°fl ^ 33€ ^-tf*

22. ^121^ 5U<H>H, ^±%% #<d3°)fe ^7) *#2] qo]S: *

23. *U7#°fl sH>H, s. *l?l*«l&±7t ^eflEioH^ -ft-3i€ *r safe 3-1-

- 68 -

NZAS-0018751

24. ^|23^°n 5a°W. S. DNA ^«go]- p. ce pacia,
P. alcaligenes, P. pseudoalcaligens £fe P. fragi Pseudomonas spp.^1
£fe Bacillus^! -8-5.1 ^ 3-§- #3.52.3. sRf-

25. $ 3H<*)sl »l^<H*]fe DSM 4109iM L iH
"t Slfe H. lanuginosa £fe 3^ -fKH^l ^<>1^?1 3i-§- 4^03.

K46, E56, S58, G61, T64, N73, S83, 190, G91, N92, N94, D96,
L97, K98, E99, 1100,. D102, A121, E129, D167, R205, E210, K237,'
N251, 1252, D254, P256, G263, L264 £fe T267 .

26. aH^X). 56-64, 83-100 SExr 205-21H 2j*ll ^Sj^l Jf-^T *H-E

^ DSM 4109i^e] ^ a- H 1^^053

27. m&*M i- 04^01^ ajojs. 0] = ^^^ ^_g. s.^ o.^
^ ,goisj|:

K46R, D57G, S58F, G61S, D62C, T64R, S83T, I90F, G91A, N92H,
N94I, N94K, L97M, K98I, I100V, D102K, A121V, E129K, D167G,
R205K, E210W, K237M, N259W, I252L, D254W, P256T, G263A, L264Q
£fe-T267W.

28. cf-l-21 q°)s_ zms. 0)^0]^ DSM 4109S*3 ^ Slfe-
H. lanuginosa ej^afl afe 3.2] -^lafls} «Jo)sj|:

S83T+N94K+D96N

E87K+D96V

E87K+G91A+D96A

N94K+F95L+D96H

A121V+R205K+E210Q

F95C+D96N

G91S+L93V+F95C

- 69 -

NZAS-0018752

E 8 7 K+G9 1 A+D9 6R+I 1 0 0 V

E87K+G91A

S83T+E87K+Q249R

S 8 3T+E8 7K+W8 9G+G9 1 A+N9 4 K+D9 6 V
N73D+S85T+E87K+G91A+N94K+D94A
E87K+G91A+L93T+N94K+D96A
D167G+E210V

N73D+E87K+G91A+N94I+D96G

S83T+E87K+G91A+N92H+N94K+D96M

E210W

E5 6T+D57L+I90F+D9 6L+E99K

E56R+D57L+V60M+D62N+S83T+D96P+D102E

D57G+N94K+K96L+L97M

E87K+G91A+D96R+I100V+E129K+K237M+I252L+P256T+G263A+L264Q

E56R+D57G+S58F+D62C+T64R+E87G+G91A+F9SL+D96P+K9ffI+K237M'

K46R, E56R,G61S

D102K

D167G

N73D+E87K+G91A+N94I+D96G

E210V

E210W

N 2 5 1W+D2 5 4 W+T2 6 7 W
S83T+E87K+G91A+N92H+N94K+D96M
E5 6R+I9 0F+D9 6L+E9 9 K
D57G+N94K+D96L+L97M.

29. *\] tfl^l 23*3-^3 °]± *<M1 a 0 v«joj ^7ji( c )oj|A^ ^asj^s ^M)^

^ sl ^

DNA ^^5. o]^*Jfe DNA

30. >|24* ^fl*l 28***1 *cW H. lanuginosa ^sMI «!<>l^i tfiS*
*Kr DNA t 1 ^*.

31. ^J29^ Sfe 30*J-°fl 4€- DNA ^3hf-£ £H*Rf

32. mi*M sa°W, f-e^ls. ^efle]£3M?I ^SlS. ^fe

- 70 -

NZAS-0018753

33. SEfe 32*<H1 3&°]H. 2- ^ -§-<*!! £4: e| ^ojsj^ sj-g-^ DNA
*\<&4: D) £$3^ g$ 31-1: ^ ^Ei

34. *fl29 s J- 30^°fl DNA £fe ^131^- 33*<Hr^ ^g.

35. 434*J°fl ^a], >4£ol 5}-g: f^Jo S 3^

36. *935*8-°fl SW*! 2-^- *M|zH 2^ >H|i?l 5J-§- ^HJAiS. *Kr

37. *a36*cN SH-M, A. niger, A. oryzae 5 A. nidulans Aspergillus
SE.fe- S. cereviciae Saccharomyces 4|s.<?l ^-t- ^JAS. t}^ 43..

38. ^36tH Si^^l, ^.^-^sl «H|b1<>} ^ °fl-&#<H,

Bacillus subtilis, Bacillus licheniformis , Bacillus lentus.
Bacillus brevis, Bacillus stearothermophilus , . Bacillus
alkalophilus , Bacillus amyloliquefaciens, Bacillus coagulans,
Bacillus circulans, Bacillus lautus , Bacillus thuringiensis 3ife
Streptomyces lividans <E^- Streptomyces murinus

Bacillus E. coli £-8- ^-g-^sl ^Eflejo)- *fli°]

°l-¥-°l*lfe. £ ^l^^sHi^ ojssM tflSH 3^

40. £<>)*fl* al^-a^^H-M >|34tf ^ 38^3 o]^ *j-ofl 4=.

. 2. ^1^^*U5l^4 £#°fl cflajj 5)^3 4^14 >HN -ygefl

41. °J21S. «l-g-^ 43. oJ i*a itSlfi $.±2) ^$3., 3fl24* Ml^l

- 71 -

NZAS-0018754

42. floH. g# $.± 0.02-200mg-§r f-8-*Hr 4^.2.3.

43. *l|41^ iEfe 42^ 4^^. *|>3-<MtM|, ^Efuf^, s)4>t||
SJ/ife €1-^ 5Ecf€- lit 4?}! i^fe 5! a^)o S ^ ^

- 72 -

NZAS-0018755

TRANSLATION

AMENDMENT

Filed by

Name

Novo Nordisk A/S

Relation
to the case

Applicant Nationality Denmark

Address

Novo Alle

DK-2880 Bagsvaerd, Denmark

Attorney

Y. S. Chang

Description
of the case

Korean Patent Application No. 96-704602

Title of
Invention

A Method of Preparing a Variant of a Lipolytic Enzyme

Date notified

August 23, 1996

Deadline

October 23, 1996

Object to
be amended

The executed Power of Attorney

Contents of
Amendment

-As per attached sheets

We are filing this Amendment pursuant to Article 13 of
the Enforcement Regulation of the Korean Patent Law.

Date : Sept. 3, 1996

Y. S. Chang
Patent Attorney

To Commissioner

Korean Industrial Property Office

Attached
Document

1 . The executed Power of Attorney with its Translation Each 1 copy

NZAS-0018756

TRANSLATION

Official Filing Charges

Amendment

W 8,000

Additional Request for Examination

Others

Total

W 8,000

NZAS-0018757

3 =r

-3 4 S

^ >H

A}^

£34 * -g- 4. 3 *L

K020, K184

4^14 7j-^ 824-20 (^SJ-tyj: : 556-8224 -6)

1996*d ^1704602 J:

1996\! 8€ 22°J

■#4(£fe^ ! 3): : §-4» , £-S- , £*Kl996. 8. 23.) *9#°}#«i):4(i996. 10. 23.)

a. ^ § 4 *J-

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1 9 9 6 -d 9€ 3«a

£ ^ S.

8,000^

1. -MS

2. $1<3# *

^3^

5,

8,000^

TEL 556-8224-6

NZAS-0018758

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